Year: 2023
Type: dissertation
Abstract: Type 2 diabetes (T2D) is characterized by elevated blood glucose and insulin in circulation, and dysregulated skeletal muscle metabolism. At the nexus of control of skeletal muscle lies the protein kinase known as mTOR. When active, skeletal muscle is anabolic, often at the expense of insulin sensitivity. In fact, skeletal muscle of rodents with T2D have elevated mTOR activity. Paradoxically, the elevated levels of anabolic activity do not lead to increased muscle mass with T2D, suggesting that mTOR’s control of autophagy has been lost under these conditions. Therefore, the purposes of this dissertation were first, to characterize myotubes from commercially available myoblasts harvested from healthy and type 2 diabetic donors; second, to describe anabolic and autophagic contributions to FSR of diabetic myotubes; and third, to modulate protein metabolism of skeletal myotubes in vitro using CRISPR-Cas9 gene editing. Results: FSR (%/h) of diabetic myotubes was significantly greater than healthy myotubes in the low (1.55 ± 0.03; 0.85 ± 0.01) mid (1.83 ± 0.08; 1.15 ± 0.05) and high (1.68 ± 0.11; 1.04 ± 0.04) glucose groups. Protein content of autophagic markers LC3B I and LC3B II was significantly greater in the healthy myotubes in high glucose media compared to diabetic myotubes in the same media. Inhibition of mTORC1, mTORC1&2, or inhibition of autophagy did not affect the FSR of healthy myotubes in euglycemic media. Additionally, inhibition of mTORC1, or mTORC1&2 did not affect FSR of diabetic myotubes in euglycemic media. However, inhibition of autophagy significantly decreased FSR of diabetic myotubes compared to diabetic controls. Finally, DEPTOR over-expression (DEPOVX) resulted in significantly lower FSR of C2C12 myotubes compared to red fluorescent protein (RFP) control myotubes; and electrical pulse stimulation (EPS) and insulin were able to affect FSR of both RFP and DEPOVX myotubes. In conclusion, diabetic myotubes present with dysregulated protein metabolism compared to healthy myotubes, and it appears that autophagy is a significant contributor to elevated FSR of diabetic myotubes. Finally, DEPTOR over-expression is feasible avenue for restoring anabolic control that still responds to canonical activation.
Author: Uranga, Selina
Language: English
Subfield: Nutrition and Dietetics
Field: Nursing
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W7056201690